Heterogeneity in CBF beta/MYH11 fusion messages encoded by the inv(16)(p13q22) and the t(16;16)(p13;q22) in acute myelogenous leukemia

Abstract

Inv(16)(p13q22) is one of the most frequent chromosomal rearrangements found in acute myelogenous leukemia (AML), representing approximately 16% of documented karyotypic abnormalities. The inv(16) breakpoints have been cloned and shown to involve the non-DNA binding component of the AML-1 transcription factor complex termed core binding factor beta gene (CBF beta) on 16q and the smooth muscle myosin heavy chain gene (MYH11) on 16p. In this study, we analyzed 37 cases of inv(16)-containing AML and 4 cases with t(16;16)(p13;q22) for expression of the CBF beta/MYH11 chimeric message by reverse transcription-polymerase chain reaction (PCR) analysis. CBF beta/MYH11 chimeric messages were detected in 33 of 37 cases with the inv(16) and in the 4 t(16;16)-containing cases. Sequence analysis of PCR products showed extensive breakpoint heterogeneity in both CBF beta and MYH11. In addition to the previously described breakpoint in CBF beta at nucleotide (nt) 495 (amino acid 165), we have identified a second novel fusion point at nt 399 (amino acid 133) in 7% of the cases. Similarly, a unique breakpoint site was identified in MYH11 at nt 1098, as well as at three previously characterized sites at nts 994, 1201, and 1921. Of the 4 PCR-negative cases, 2 of 3 tested lacked CBF beta rearrangements by Southern blot analysis, suggesting the possible involvement of a different genomic locus in some cases with cytogenetic evidence of inv(16). To assess whether the portions of CBF-beta contained within the CBF beta/MYH11 chimeric products retain the ability to interact with their heterodimeric DNA-binding partner AML-1, we performed in vitro DNA- binding analysis. Recombinant CBF-beta polypeptides consisting of the N-terminal 165 amino acids retained their ability to interact with AML-1, whereas mutants containing only the N-terminal 133 amino acids interacted with AML-1 less efficiently. These data suggest that different CBF beta/MYH11 products may vary subtly in their biologic activities.

Frequency of CBFβ/MYH11 fusion transcripts in patients entered into the U.K. MRC AML trials

“…PCR products were electrophoresed through low melting point agarose, the required band extracted using Wizard TM PCR preps (Promega). These were sequenced using the fmol TM DNA Sequencing System (Promega) with an end-labelled primer S1 (5 0 -TTAGCACAA-CAGGCCTTTGAA-3 0 ), derived from the CBFb portion of the fusion transcript (Shurtleff et al, 1995).…”

Underestimation of inversion (16) in acute myeloid leukaemia using standard cytogenetics as compared with polymerase chain reaction: results of a prospective investigation

“…In one patient (no. 9), a novel breakpoint was detected within the CBFb gene (Shurtleff et al, 1995). The table displays the morphological as well as the cytogenetic and molecular findings.…”

“…In nine patients both karyotypic and molecular analysis could be compared. In six of these nine patients cytogenetics revealed inv (16) (Shurtleff et al, 1995). Lane 12 represents a negative control.…”

Pathogenesis of acute myeloid leukaemia and inv(16)(p13;q22): a paradigm for understanding leukaemogenesis?

“…reaction (RT-PCR) (Claxton et al, 1994;Hébert et al, 1994;Poirel et al, 1995), Southern blot analysis (Van der Reijden et al, 1995a) and two-colour interphase fluorescence in situ hydridization . To date, 10 differently sized CBFb-MYH11 transcripts (Fig 1), have been identified, types A-H Shurtleff et al, 1995;Van der Reijden et al, 1995b;Costello et al, 1997a), type I or S (Dissing et al, 1998;Grardel et al, 2002;Van der Reijden et al, 2001) and type J (Springall et al, 1998;Trnkova et al, 2003). The majority (85%) of cases of inv (16), or t(16;16), are associated with the type A fusion transcript, corresponding to an in-frame CBFb nt 495-MYH11 nt 1921 junction , while transcript types B-J are rare.…”